The present invention generally relates to protein crystals and methods for identifying diffraction-quality protein crystals.
In proteins, structure dictates function. The major cost in terms of time and expense in protein structure determination by x-ray crystallography often rests in the identification of conditions for generating diffraction-quality protein crystals. With the human genome now sequenced, efforts over the last decade have shifted increasingly toward structural characterization of the proteins encoded by the genome, primarily through diffraction analysis. Major bottlenecks in this effort rest in the time and expense associated with isolating and purifying functional proteins, and identifying appropriate conditions for growth of diffraction-quality crystals. Several high-throughput platforms for screening protein crystallization conditions have been developed with reasonably good success. (Cumbaa, C. A.; Lauricella, A.; Fehrman, N.; Ceatch, C.; Collins, R. W.; Luft, J.; DeTitta, G.; Jurisica, I., Automatic classification of sub-microlitre protein-crystallization trials in 1536-well plates. Acta Crystallographica D 2003, D59, 1619-1627. Spraggon, G.; Lesley, S. A.; Kreusch, A.; Priestle, J. P., Computational analysis of crystallization trials. Acta Crystallographica D 2002, D58, 1915-1923. Echalier, A.; Glazer, R. L.; Fülöp, B.; Deday, M. A., Assessing crystallization droplets using birefringence. Acta Crystallographica D 2004, D60, 696-702. Bodenstaff, E. R.; Hoedemaeker, F. J.; Kuil, M. E.; de Vrind, H. P. M.; Abrahams, J. P., The prospects of protein nanocrystallography. Acta Crystallographica D 2002, D58, 1901-1906. Blundell, T. L.; Jhoti, H.; Abell, C., High-Throughput Crystallography for Lead Discovery in Drug Design. Nature Reviews Drug Discovery 2002, 1, 45-54. Zheng, B.; Roach, L. S.; Ismagilov, R. F., Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets. J. Am. Chem. Soc. 2003, 125, 11170-11171. Santarsiero, B. D.; Yegian, D. T.; Lee, C. C.; Spraggon, G.; Gu, J.; Scheibe, D.; Uber, D. C.; Cornell, E. W.; Nordmeyer, R. A.; Kolbe, W. F.; Jin, J.; Jones, A. L.; Jaklevic, J. M.; Shchultz, P. G.; Stevens, R. C., An approach to rapid protein crystallization using nanodroplets. J. Appl. Crystallography 2002, 35, 278-281.) However, the number of conditions that can be sampled in any screening measurement is ultimately dictated by the total quantity of initial purified protein and the smallest detectable protein crystal. Reduction in the detection limits for protein crystallization can potentially reduce both the time required for performing an assay of conditions and the total amount of protein consumed. The challenges associated with early detection of protein crystallization are numerous. Optical detection approaches are arguably most directly compatible with diverse crystallization platforms and enable continuous monitoring of the same sample at multiple time-points. Commercially available approaches based on image analysis (Cumbaa, C. A.; Lauricella, A.; Fehrman, N.; Ceatch, C.; Collins, R. W.; Luft, J.; DeTitta, G.; Jurisica, I., Automatic classification of sub-microlitre protein-crystallization trials in 1536-well plates. Acta Crystallographica D 2003, D59, 1619-1627. Spraggon, G.; Lesley, S. A.; Kreusch, A.; Priestle, J. P., Computational analysis of crystallization trials. Acta Crystallographica D 2002, D58, 1915-1923) or birefringence (Echalier, A.; Glazer, R. L.; Fülöp, B.; Deday, M. A., Assessing crystallization droplets using birefringence. Acta Crystallographica D 2004, D60, 696-702) are limited to crystals with dimensions spanning at least several μm. Incorporating a fluorophore by doping (Groves, M. R.; Müller, I. B.; Kreplin, X.; Müller-Dieckmann, J., A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye. Acta Crystallographica D 2007, D63, 526-535.) or covalent attachment (Forsythe, E.; Achari, A.; Pusey, M. L., Trace fluorescent labeling for high-throughput crystallography. Acta Crystallographica D 2006, D62, 339-346.) can improve on these detection limits, but also introduces a significant background signal from solvated dye molecules and amorphous aggregates.
All of these previously established methods suffer from the inability to easily detect sub-diffraction limited crystals and to discriminate between the formation of protein crystals versus the localized deposition of amorphous protein aggregates. What is needed is a sensitive and selective detection method for protein crystals and crystallization, with detection limits for the onset of crystallization corresponding to crystal dimensions well below the optical diffraction-limit.